Microvillus Inclusion Disease (MVID) is a life-threatening pediatric disease that results in severe intractable secretory diarrhea from the first days of life. MVID-affected infants and children do not survive unless they are either supported continuously by intravenous parenteral nutrition, or undergo small bowel transplantation. Eight years before loss of function of Myosin Vb (Myo5b) was identified as central to MVID pathogenesis, Ameen and Salas provided the first demonstration of an apical trafficking defect of the major solute transporters (CFTR, NHE3) in MVID patients intestinal brush border. Myo5b is an unconventional myosin that associates with apical recycling endosomes (ARE) by binding to small GTPases such as Rab11a to regulate trafficking and movement of non-raft associated cargo from the ARE to the apical plasma membrane. How mutations in Myo5b lead to defects in apical brush border membrane, the development of microvillus inclusions in enterocytes, apical trafficking defects of raft associated transporters and massive diarrhea in MVID patients is unknown. No whole animal model of MVID has been established to date. KO animals in genes other than Myo5b display similar subcellular phenotypes, but no diarrhea. Our published and preliminary data in MVID patient samples and polarized intestinal CaCo-2BBe cells constitutively depleted in Myo5b show (i) loss of apical exocyst complex component Sec8, which is involved in raft-mediated NHE-3 insertion in the apical membrane; (ii) a profound delocalization of PDK1 and its activation target, atypical PKC; and (iii) rescue of water absorption by a PDK1 inhibitor. These previously unsuspected changes in traffic and signaling may be critical because they are potentially druggable. The combined expertise in brush border myosin trafficking of apical ion transporters in the Ameen lab and the Salas laboratory's expertise in signaling will be harnessed to unravel the molecular mechanisms affected downstream of the Myo5b mutation. This knowledge will allow development of pharmacological therapeutic targets to ameliorate MVID diarrhea. This proposal will test the hypothesis that loss of function mutations in Myo5b result in dysfunctional membrane traffic and signaling (PDK1, Akt, aPKC, SGT1) causing changes in insertion and activation of multiple ion transporters (including but not restricted to NHE3). This results in decreased absorption and increased secretion, and ultimately in secretory diarrhea. The experiments will be carried out in transduced CaCo-2BBe cells that mimic enterocyte physiology and MVID pathology under Myo5b knockdown. This model is a refinement of previous MVID models in culture developed in Ameen and Salas laboratories which, unlike cell lines previously reported, can be used for water transport experiments. The results will be validated in de-identified MVID samples, non-transformed human colon epithelial cells in primary culture, and a novel Myo5b knockdown zebrafish model that displays increased gut water content under Myo5b knockdown. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page